Method for promoting wound healing by administering a compound which binds ldl-receptor-related protein (lrp) ligand binding domain

ABSTRACT

The present invention relates to the field of therapeutic methods, compositions and uses thereof, that affect, directly or indirectly, the behavior of LRP receptors. These compositions and methods result in the treatment of inflammatory, immunological and metabolic conditions. More particularly, the methods and compositions of the invention are directed to the identification of small molecules, drugs and/or pharmacological agents that affect the Wnt pathway by affecting normal complex formation among various signaling receptors, the LRP5 and LRP6 receptor, and related ligands.

REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Continuation of Ser. No. 14/680,547, filed Apr. 7,2015, which is a Divisional of application Ser. No. 12/221,863, filedAug. 7, 2008 (now U.S. Pat. No. 9,046,537), which is aContinuation-in-Part of application Ser. No. 11/598,916, filed Nov. 14,2006 (now U.S. Pat. No. 8,367,822), which is a Continuation-in-Part ofapplication Ser. No. 11/097,518, filed Apr. 1,2005 (abandoned), which isa Continuation-in-Part of application Ser. No. 11/084,668, filed Mar.18, 2005 (now U.S. Pat. No. 8,461,155), which is a Continuation-in-Partof application Ser. No. 10/849,067, filed May 19, 2004 (now U.S. Pat.No. 8,637,506), which claims the benefit of U.S. Provisional PatentApplication No. 60/504,860, filed Sep. 22, 2003, each of which is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Receptors which are binding sites for proteins and small molecules areattractive targets for pharmacological intervention in disease-relatedprocesses. One group that fits this category of receptors is comprisedof members of the LRP family. The term LRP is an abbreviation forLDL-Receptor-related Proteins, where the LDL receptors are a group ofproteins involved in the binding and transportation of Low-densityLipoprotein (LDL) into cells by endocytosis. Various proteins areconsidered to be members of the LRP family because of their resemblanceto LDL-receptors as well as their resemblance to each other. FIG. 1shows various members of the LRP family, where different motifs that areheld in common are shown for various members. The most important commonelements are the YWTD β-propellers, EGF-like domains and LDLreceptor-like ligand binding domains. These elements may appear assingular elements or they may comprise multimeric repeats. The membersof this family are also characterized by a transmembrane domain thatanchors the LRP extracellular portion to a membrane surface as well asan intracellular domain that may interact with cellular proteins.Although the LRP family members are structurally related, the functionsthey serve in vivo are of a diverse nature that include the uptake oflipoproteins, endocytosis, transcytosis, signal transduction, vitaminand hormonal homeostasis, as well as phagocytosis of necrotic cells(reviewed in Herz and Strickland 2001 J. Clin. Invest. 108:779-784). Inconjunction with the various roles that these proteins may be involvedin, members of the LRP family recognize a large number of ligands. Forinstance, one member alone, LRPI, recognizes at least. 30 differentligands that in themselves represent several families of proteins. Theseligands include lipoproteins, proteinases, proteinase inhibitorcomplexes, ECM proteins, bacterial toxins, viruses, and variousintracellular proteins.

Some of the proteins that bind to members of the LRP family are involvedin Wnt signaling. For example, Wnt has been shown to directly interactwith one or more of the YWTD domains of the amino (extracellular)portion of LRP5 and LRP6 to induce Wnt signaling. Another example isDkk, which is believed to bind to different domains of LRP5 and LRP6(the third and fourth YWTD domains) but nonetheless influences theability of the first or second domain of LRP5 and LRP6 to bind to Wnt.Other proteins such as Frat 1 (Hay et al. 2005 J Biol Chem 14;13,616-13,623), Christin/R-spondin proteins (Nam et al., JBC 2006) andconnective-tissue growth factor (CTGF) (Mercurio et al., 2003Development 131; 2137-2147) also interact with the extracellular Domainsof LRP5 while Casein kinase I (Davidson et al. 2005 Nature 438; 867-872,Swiatek et al., 2006 J Biol Chem 281; 12,233-12,241), Glycogen synthasekinase 3 (GSK3) (Mi et al., JBC 281; 4787-4794, Zeng et al., Nature 438;873-877) and Axin (Mao et al., 2001 Mol Cell 7; 801-809) have been shownto interact with the intracellular portion. The ability to bind to aprotein may or may not be involved in signal functions of an LRPmolecule. For example, the majority of ligands that bind to themultiligand receptor LRP1 are either proteases or molecules associatedwith the control of proteolytic activity. However, although the LRP 1receptor is not commonly associated with Wnt pathway events,investigations have revealed that under appropriate conditions,truncated versions of LRP1 were able to interact with Frizzled, a majorcomponent of the Wnt signaling pathway (Zilberberg et al., 2004 J. Biol.Chem. 279; 17,535-17,542). This interaction is dissimilar to the wellcharacterized system involving interactions of LRP5 and LRP6 and Wntelements since the effect of both the truncated as well as the fulllength version of LRP 1 is the opposite of the classical LRP5 and LRP6interactions. The binding of LRPI to Frizzled represses Wnt signalinginstead of inducing it.

Some of the proteins that bind to members of the LRP family are notinvolved in Wnt signaling. Even with LRP members like LRP5 and LRP6,which are known to play a major part in Wnt signaling, certain ligandsthat bind to LRP5 and LRP6 have been shown not to affect the Wntpathway. For instance, Wei et al. have demonstrated that LRP6 mediatesthe internalization and lethality of anthrax toxin (Cell 124, 1141-1154,Mar. 24, 2006), and the role of LRP5 in cholesterol metabolism isbelieved to be Wnt independent (Magoori et al., 2003 J. Biol. Chem. 278;11,331-11,336). With regard to the latter, Fujino et al. (2003 Proc.Nat. Acad. Sci. (USA) 100; 229-234) investigated the metabolicconsequences of a genetic ablation of LRP5 and concluded that LRP5 isessential for both normal cholesterol metabolism and glucose-inducedinsulin secretion. The presence of an LRP5 deficiency in eitherhomozygous (LRP5 −/−) or even heterozygous (LRP5 +/−) mice resulted in asignificant increase in plasma cholesterol levels when the animals werefed a high-fat diet. Although fasted blood glucose and insulin levelswere normal in the mutant strains, they showed a defect in glucosetolerance when challenged. These animals also showed impaired clearanceof chylomicron remnants and also impaired glucose-induced insulinsecretion from the pancreatic islets. The effect of a lack of LRP5 wasalso tested in a double mutation situation where the mice lacked notonly LRP5, but also apoE (Magoori et al. 2003). Although neithercondition alone led to changes in cholesterol levels with a normal diet,the double condition led to 60% higher plasma cholesterol levels. At 6months of age, the double-null mice had also developed severeatherosclerotic lesions that were three times larger than those inknockout mice missing only apoE. The connection between LRP moleculesand metabolism is also evidenced by the discovery that certainpolymorphisms in the LRP5 gene have been correlated with obesityphenotypes in a family based study (Guo et al., 2006 J. Med. Genet. 43;798-803). Lastly, a mutation in LRP6 has been correlated to an autosomaldominant defect that results in the expression of phenotypic featuresassociated with metabolic syndrome: hyperlipidemia, hypertension anddiabetes (Mani et al., 2007 Science 315; 1278-1282).

There is a distinction between transducer (LRP5 and LRP6 receptors) andnon-transducer multi-ligand receptors (non-LRP5 and non-LRP6 receptors).In the case of a non-transducing receptor, the term “multi-ligand”encompasses broad specificity, as in the case of a receptor that takesup different monosaccharides. In this case, essentially the same effect(transport) is carried out by the receptor for a variety of differentligands where each internalized ligand is then recognized and processedaccording to its specific chemical nature. On the other hand, formulti-ligand signal receptors, another layer of complexity is observedwhere different domains participate in different reactions. In the caseof signal transducers, the ligand per se is not the target of furtherdownstream actions. In fact, as a rule, it is not even internalized.Thus, the specificity of the signal transduction is entirely the resultof the specificity of the transducer. This means that if two differentligands elicit two different downstream responses, there must be adifference, however subtle, in the way they trigger the transducer afterbinding.

With regard to the LRP5 and LRP6 receptor, it is quite obvious that theextracellular and intracellular domains must by necessity have differentligands and different functions. Even within the extracellular portionitself, there will be differentiation of function for the differentdomains of LRP5 and LRP6. For example, the first two YWTD domains in theextracellular portion of LRP5 and LRP6 are involved in binding Wnt andtransmitting a signal, while the third and fourth domains are sites forbinding of a completely different protein, Dkk, and a subsequentdampening of Wnt signaling. Remarkably, LRPs combine features of bothtypes of multi-ligand receptors since they can function both as aninternalizer and as a transducer.

Although domains of functional and structural similarity can beidentified through amino acid alignments, the ability of such analoguesto carry out different functions is a product of their fine differences.As described in the review article by Herz and Stickland that was citedearlier: “Crystallographic and nuclear magnetic resonance studies ofindividual repeats have revealed that the sequence variability in shortloop regions of each repeat results in a unique surface contour surfaceand charge density for each repeat.” In summary, even when a collectionof repeated sequences are able to form similar structures, theparticular nature of the amino acids on their exposed surfaces willstill dictate the ability to bind different ligands. Interactionsbetween individual amino acids will also cause differences in theoverall structure where cavities in comparable domains may be slightlylarger or smaller due to small scale attractive or repulsive forces.This can be seen in the studies of LRP5 and LRP6 where the size of theopening in the β-propeller of a YWTD repeat region is different from onedomain to another. More importantly, as described in section 4.2 of U.S.Patent Publication No. 2005/0196349, identification of amino acidresidues that are important for Dkk binding was carried out by alaninescanning. A comparison of nucleic acid and amino acid sequences showsthat there are substitutions of different amino acids at analogous sites(U.S. patent application Ser. No. 11/598,916) within these cavitiesthereby differentiating the degree of affinity between molecules thatmay be similar in size but different in terms of polarity and/or chargewith regard to binding to each of the domains.

In the previously cited patent applications, the use of a detailedthree-dimensional model of the LRP5 receptor allowed a virtual screeningof a library of compounds for predicting molecules that would fit into abinding domain ofLRP5. As disclosed in U.S. Patent Publication No.2005/0196349, a variety of different biological results can be seen whenthese compounds are tested with in vitro assays. Looking at Table II, itcan be seen that some of the compounds (Group 1) are toxic asexemplified by compounds IIC5, IIIC6, and IIIC12 which reduced basalexpression to 26%, 0% and 10%, respectively. Not surprisingly, furtherexperiments showed a lack of stimulation when Wnt was added. Othercompounds such as IIC6, IIC18 and IIC19 were not intrinsically toxic,since they maintained or even stimulated basal level expression.However, in this group of compounds (Group 2), the addition of Wntshowed no stimulation, indicating an inability to respond to Wnt in thepresence of these compounds. A third class of compounds (Group 3) showeda normal level of response to the addition of Wnt compared to the nodrug control, but showed a diminished effect of inhibition by Dkk. Forinstance, IIC8 (NCI 39914) allowed essentially the same level ofstimulation by Wnt as in its absence (1227 compared to 1000 in theabsence of drug), but when Dkk was added, the amount of activity wasonly reduced to 476. The control showed a shift of 1000 to 100 by theaddition of Dkk. Even more strikingly, IIIC3 (NCI 8642) shows almost thesame amount of activity in the presence of Dkk as in its absence,demonstrating that the binding of this molecule can lead to a block inWnt suppression by Dkk. There is even one compound, IIC9, thatrepresents a fourth group of compounds that was able to reduce theamount of Wnt stimulation, but instead of showing Dkk suppression, Wntactivity was stimulated three fold by the presence of Dkk. Thus, it canbe seen that binding to LRP5 and LRP6 does not necessarily lead to asingle phenotype in these assays.

There are a variety of reasons why these different effects may be seen.For instance, although one particular domain was chosen for theselection of a ligand from the library, a biological assay may revealthat the affinity of the compound is higher for a different (butsimilar) domain on the target protein. There is also the possibility ofmimicry, where the binding of the compound to the Dkk site on LRP5 andLRP6 in itself emulates the same effect seen by binding of the trueligand and leads to “Dkk-like” suppression of Wnt activity in theabsence of Dkk. It is also natural to assume, especially in the case ofa multi-ligand receptor, that allosteric effects are possible thatinfluence separate binding events at sites away from where the drugitself may bind.

In the previously disclosed applications, molecules with propertiesdescribed for the third class of compounds (Group 3) were tested forvarious biological activities besides the LEF reporter system in orderto test for a biological effectiveness for disease processes. Among theassays described in these disclosures were those related to boneformation and remodeling as witnessed by assays for osteoblastdifferentiation in U.S. Patent Publication No. 2005/0196349. Twocompounds from this group, IIC8 and IIIC3, were tested for an additionalproperty, the ability to block the binding of sclerostin, a proteinwhich has previously been shown to have an effect similar to that of Dkkin being able to block Wnt signaling. Experimental results showed adirect correlation where increased amounts of these compounds resultedin decreased binding of sclerostin-AP. These compounds as well as othersimilar compounds were also tested for effects on bone growth viacalavarial bone formation, β-catenin activity and viability in varioustumor cell lines, tumor induction in a mouse model, as well as metaboliceffects such as cholesterol and glucose metabolism (U.S. patentapplication Ser. No. 11/598,916). The potential use of pharmaceuticalcompositions for altering the activity of LRP5 in a subject has beendescribed in U.S. Patent Publication No. 2003/0181660 (herebyincorporated by reference) with specific application to diseases such asdiabetes, autoimmune diseases, viral infections, osteoporosis andmetabolic disorders, as well as diseases that involve or affectendocytosis, antigen presentation, cytokine clearance or inflammation.However, their approach was directed towards a different level, wherethey taught the use of compounds to regulate the level of expression ofLRP5. In contrast, the methods described in U.S. Patent Publication No.2005/0196349 have been directed towards the identification of compoundsthat interact with the LRP5 and LRP6 protein or associated proteins.

A similar program of virtual screening followed by binding studies wascarried out for compounds predicted to bind to Disheveled, anothermember of the Wnt signaling pathway (U.S. patent application Ser. No.11/097,518). In this case, molecules of interest were followed withtesting for effects on embryogenesis.

SUMMARY OF THE INVENTION

The present invention discloses the identification and use of moleculesthat bind to members of the LRP family thereby providing for relief insubjects suffering from inflammation, an immune mediated disorder, ametabolic disorder, a pathological condition associated with anelevation of TNF-α, a pathological condition associated with elevationof mmp, a skin condition or disease, an organ or tissue injury or anycombination of the foregoing. Other molecules that may be of use in thepresent invention may bind to a factor that interacts with an LRPthereby preventing its binding to LRP, where the disruption of thisbinding may also provide relief from the foregoing conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings.

FIG. 1 is a diagram of the structure of various members of the LRPfamily showing the location of various motifs (taken from FIG. 2 of Heet al. “LDL Receptor-Related Proteins 5 and 6 in Wnt/b-cateninSignaling: Arrows Point the Way” 2005 Development 131; 1663-1677).

FIGS. 2A and 2B are graphs showing the effects of various concentrationsof Gallic Acid (Panel A) and Digallic Acid (Panel B) upon Wnt activityand the suppression of Wnt activity by Dkk.

FIGS. 3A and 3B are graphs showing the effects of lower concentrationsof Gallic Acid (Panel A) and Digallic Acid (Panel B) upon Wnt activityand the suppression of Wnt activity by Dkk.

FIG. 4 is a picture of the effects of IIC8 on new bone formation.

FIGS. 5A and 5B are pictures (Panel A) and a graph (Panel B) relating tobone loss in periodontitis and the effectiveness of compound IIC8 in itstreatment. Panel A are pictures showing protection against bone loss inLPS-induced periodontitis with macroscopic images of jaws. Arrowsindicate the sites between the first and second molars where LPS orcontrol saline was injected. View I is a front view and View II is a topview. Panel B is a graph showing measurements of average distancesbetween Cemento-enamel junctions (CEJ) and alveolar crests, indicatingthe degree of bone loss in a periodontal model.

FIGS. 6A and 6B are a graphs showing the effects of Dkk mutations onTNFα parameters (Panel A) and blood glucose levels (Panel B).

FIG. 7 is a graph showing the effects of Dkk(−/−) mutation onAdiponectin levels.

DETAILED DESCRIPTION OF THE INVENTION

In the previously cited U.S. Patent Publication No. 2005/0196349, amethodology was disclosed that was successful in identifyingpharmacological agents that can influence Wnt activity in a subject.Various procedures, including mutational analysis, alanine scanning,crystallography, NMR spectroscopy, homology modeling, and threedimensional models of target proteins involved in the Wnt pathway, wereall used for virtual screening of a library of compounds to selectcompounds capable of binding to selected portions of targets involved inprotein-protein interactions.

By binding to these elements, the present invention discloses thatbeneficial effects may be induced either by influencing Wnt signaling orby taking advantage of non-Wnt signaling effects that are alsoproperties of the LRP signal receptors. In many cases, differentiationbetween these signaling routes is not necessary, since only the neteffect may be of interest rather than the particular mechanism. Thus,when carrying out screening assays, the particular effect may beascertained for a molecule based upon effects on a marker for the Wntpathway, or a biological assay may be carried out that does not directlymonitor Wnt signaling and serves as a marker for only the desiredresult. As an example of the latter, the ability to alter the amount ofTNF-α in a subject can be an effect-oriented assay that measures theamount of TNF-α secreted by cells in the presence of a molecule that isbeing tested for pharmaceutical efficacy. In a similar fashion, any of avariety of animal models that are used for induction of inflammatoryresponses may be used for testing of effects by molecules that have beenselected on the basis of being able to bind to LRP or to one of theelements that interact with LRP.

In U.S. Patent Publication No. 2005/0196349 (“the '349 Publication”),virtual screening allowed the selection of a number of molecules thatwere subsequently tested for their ability to bind to LRP5 and LRP6.Success in this approach was seen by the high number of molecules fromthis screen that were able to affect the binding of aalkaline-phosphatase labeled Dkk molecules to full length LRP5 (seeTable I of the '349 Publication). A variety of effects were seen wheresome compounds induced an inhibitory effect of the binding of thelabeled Dkk to LRP and other compounds were actually able to induce anincreased level of binding. In a further step, the molecules were testedwith a biological assay for an ability to inhibit a Wnt-mediated assay.Therefore, in Tables II and III of the '349 Publication, moleculesselected for an affinity for the Dkk binding site of Domain III of LRP5had various effects on Wnt activity, where some showed no effects, someincreased Wnt activity and some showed decreased Wnt activity. In asecond biological assay, Wnt activity was also measured in the presenceof Dkk, a repressor of Wnt activity. In this particular application,only molecules that lacked effects upon Wnt activity but were able toalleviate Dkk suppression of Wnt activity were used as model moleculesfor a further screening step. However, although this particularbiological assay was applied, that the ability of a molecule to bind toLRP6 may provide therapeutical benefits should not be ruled out becauseof an inability to negate Dkk-mediated Wnt suppression.

The complexity of the Wnt system can also be seen in FIG. 21 of U.S.patent application Ser. No. 11/598,916 where a dose dependency curveshowed differential effects: at low dosages, the binding of ENZO MO1 Dkkblocked repression of Wnt signaling and it declined as the dosage wasincreased. However, at higher concentrations there is a reversal of thiseffect and with increasing dosages there was an actual increase in Wntactivity. As previously disclosed in U.S. patent application Ser. No.11/598,916, because of the similarity of the domains in LRP5 and LRP6,the selection of an agent for binding to one domain may also be aselection of an agent that has affinity for an unselected, but similardomain. This would be especially true for another domain on the LRP5 andLRP6 receptor but as mentioned earlier, analogous YWTD Domains arepresent on other members of the LRP family as well. With reference tomulti-targeting of LRP5 Domains, modeling experiments with the predictedstructure of a LRP6 in conjunction with the structures of IC15 and IIIC3show that although IIIC3 shows excellent fitting within the cavity ofthe YWTD Domain III used for the virtual screening described in the '349Publication, another molecule, IC15, selected on the same basis actuallyshows a better fit with YWTD Domain II, indicating that it may have ahigher affinity for this domain rather than the one used in thescreening. A similar effect may be taking place in some examples, whereat low concentrations, YWTD Domain III is occupied by a selectedcompound, but at a higher concentration, the lower affinity targetsDomain I or II may be occupied, and thereby either decrease the amountof Wnt that can bind or otherwise hinder its ability to transmit asignal. Although the inventors have not carried out investigations ofcompounds that in the absence of Dkk either: a) knocked down Wntactivity; or b) acted as a stimulator of Wnt activity, these compoundsmay have higher affinities for Domains I and/or II rather than theDomain III structure used in the virtual screening. In the former case,the compounds may reduce Wnt activity by decreasing the amount of Wntthat can bind or interfere with Wnt signal transmission and in thelatter case, the compounds may mimic the binding of Wnt and providetheir own stimulatory signal.

As a result of the multiplicity of similar Domains on even a single LRPreceptor as well as the similarity between the various LRPs, a moleculeselected to bind to the Dkk binding Domain of LRP 6 may have a varietyof physiological effects that may or may not be associated with Dkkbinding and furthermore, these effects may or may not be associated withthe Wnt pathway. With regard to the latter case, the selection of amolecule for binding to the Dkk binding site may be viewed as using asite for binding in general. Given that these receptors are signalgenerating moieties that depend upon binding events and likelyallosteric rearrangements, a binding of a molecule to one particularsite may have profound effects on the binding at other sites as well asthe activity of the receptor for other functions that are carried out atother sites. The present invention takes advantage of the fact that anLRP receptor with a bound ligand will have altered properties comparedto a receptor without a ligand. As such, the present invention disclosesthat either one of two approaches may be appropriate afteridentification of a binding molecule. In the first approach, amechanistic methodology is employed where a particular stepwise pathwayis used in assays, where the ability of being able to relieve Dkksuppression is used as a criterion for use in Wnt activation (or moreexplicitly blockage of Dkk suppression of Wnt), as identified by asurrogate marker such as the LEF reporter gene. This was the approachtaken in the previously cited applications and it has been shown toresult in the identification of a number of useful compounds. Applicantsnow disclose that a more functional approach may also be taken thateschews mechanisms and looks at applications instead. In this approach,the ability to bind to the LRP receptor is the basis of selection, butthen direct effects upon the physiological problem are assessed ratherthan the LEF surrogate marker.

This direct approach may result in the identification of more compoundsthan might not be apparent with only the mechanistic approach. Forinstance, it is known that Dkk1 and Dkk2 have mutually antagonisticeffects such that under some circumstances Dkk1 represses Wnt activitybut Dkk2 leads to induction or enhancement (Wu et al., 2000 Curr Biol10; 1611-1614; Zorn 2001 Curr Biol 11; R592-R595; Brott and Sokol 2002Molec and Cell Biol 22; 6100-6110). Thus, when a molecule is selectedfor prevention of binding of other proteins to LRP5 and LRP6 by virtueof the structure of the Dkk binding site of LRP5 and LRP6, both Dkk1 andDkk2 interactions are potentially affected. As such, a pharmacologicalagent that binds to this site may have entirely opposite effectsdepending upon whether the activity is based upon a cellular environmentwhere binding of Dkk1 or Dkk2 is more important. As such, evaluation ofa net clinical effect may be of more importance than that of individualsteps. This will especially hold true in animal studies where numerousdifferent cell types are involved in both disease manifestation as wellas possible curative processes. The importance of cellular milieu forWnt signaling has been noted before for Dkk2 where it can act as a Wntrepressor or activator and in a paper by Mikels and Nusse (2006 PloS 4;0570-0582) where Wnt5a can either activate or inhibit a β-cateninreporter gene. Lastly, it was earlier disclosed that LRPI can affect Wntsignaling and that LRP4, another member of the LRP family has beenconsidered to be involved in the Wnt signaling system due to itssimilarity to LRP5 and LRP6 in the organization and sequences of itsextracellular domains and the effects on limb development by mutationsin the gene coding for LRP4 (Johnson et al. 2006 Genomics 88; 600-609,Simon-Chazottes et al., 2006 Genomics 87; 673-677). As such, selectionof a compound that binds to a β-propellor region of LRP5 or LRP6 mayalso be a selection for an agent that binds to other members of the LRPfamily as well with results that may affect roles that these other LRPmembers participate in that may be different from those of LRPS andLRP6.

It has been previously described in U.S. patent application Ser. No.11/598,916 that either component of a protein/protein interaction may bea candidate for pharmaceutical intervention with identified compounds(as described in U.S. patent application Ser. Nos. 10/849,643,10/849,067, 11/084,668, 11/097,518, 11/598,916, 60/963,771 and60/965,279). These compounds may include a small molecule, protein,peptide, polypeptide, cyclic molecule, heterocyclic organic molecule,nucleic acid, lipid, charged lipid, polar lipid, non-polar lipid, sugar,glycoprotein, glycolipid, lipoprotein, chemical, or a fragment of acompound that comprises a heterocyclic organic molecule, nucleic acid,lipid, charged lipid, polar lipid, non-polar lipid, sugar, glycoprotein,glycolipid, lipoprotein, or chemical. Thus, it is also a subject of thepresent invention that ligands that bind to LRP molecules may also betargets, where the same methods previously described for identifyingcompounds that bind to LRP receptors may also be applied to the ligandsthat bind directly and indirectly to LRP receptors.

Another object of the present invention is to subdivide core compoundsthat have been found to affect the selected targets into subcores thatmay also bind to the selected targets. These subcores may also be usedto identify additional effective compounds. For example, IIIC3(illustrated below) was used to identify the following core compound 1a:

Division IIIC3 results in the two following components:

which can be described as separate subcores with the followingstructures:

This reductionist approach may be exploited by virtual screening wherethe subcore is added to various other groups and tested for a predictedability to bind to the target structure. Alternatively, some empiricalexperiments may also be carried out where molecules that correspond tothese subcore regions are obtained or synthesized and tested out inappropriate bioassays. In some cases there may be similar effects to theparent core compound by the subcore compound whereas in other cases thesubcore requires additional contacts provided by other parts of theparent core molecule to provide sufficient binding ability to the targetprotein to provide a biological effect. As an illustration of thisapproach, Gallic Acid, which is a small molecule similar to the SubcoreA shown above, was tested for its effect upon Wnt activity. The resultsare described in Example 1 and shown in FIG. 2 where it can be seen thatthe a small molecule derived from the subcore of Core compound 1a(Gallic Acid) can by itself block the effects of Dkk repression.

If a subcore is sufficiently small and has been shown to have somedegree of effectiveness, it may be used to design a dimeric molecule.This dimeric molecule will be especially useful when the target regioncomprises repeated amino acid sequences such that the region thatprovides a binding site for the subcore is present in multiple copiesi.e., a first subcore of the dimer can bind to one portion of the targetwhile a second subcore of the dimer binds to its corresponding region inthe same binding area. For instance, a dimeric compound can be made andtested with either Subcore A or Subcore B shown above. An illustrationof this point is described in Example 2 and FIG. 3 where a dimericversion of Gallic Acid was tested and shown to be effective at aconcentration where Gallic Acid itself has no effect, indicating anenhanced affinity for the multimeric form compared to the monomericform. If the target regions are large enough, more than two subcores canbe joined together to bind to various repeat units of the target region.

In contrast, if the binding subdomains of the target are dissimilar, itmay be more useful to append a different chemical group to the firstsubcore to provide additional binding ability. This particularcircumstance will arise when the target region is not made up of repeatunits. It may also arise when repeated units are used for structuralcomformation but the amino acids that are exposed as contact points arethe sites where the repeat sequences diverge from each other. To use theexemplary IIIC3 molecule above, the binding affinity of the corecompound to LRP may be considered to be a summation of the bindingabilities of Subcore A (essentially a Gallic Acid moiety) to bind to onesite and the ability of Subcore B to bind to another site within thetarget region. In the absence of thermodynamic data concerning theparticular contribution from each moiety, it is unknown whether thereare similar levels of binding stabilization endowed by each moiety orwhether it may be disproportionate in nature. It is even possible thatthe observed binding from a molecule is sufficiently asymmetric thatmost of the affinity of the compound derives from one subcore and themajor contribution of the other subcore is only a neutral aspect, i.e.not interfering with the ability of the first subcore to bind. In thistype of case, there would be opportunity for obtaining a more effectivecompound, by partnering the functional subcore with a different compoundthat can more actively contribute to binding.

There are a variety of ways that a subcore may be partnered with otherchemical moieties to identify a more effective pharmaceutical agent.Reviews of such methods are summarized in Erlanson et al., 2004 (J MedChem 47; 3463-3482) and Erlanson 2006 (Curr Opin Biotech 17; 643-652)for a process termed fragment based drug discovery. This can take theform of virtual screening where various groups are appended to thesubcore and predictions on binding capability are carried out followedby biological assays similar to the way compounds were first identified.Alternatively, it may occur by the empirical testing of compounds thatcomprise the subcore, linked to other chemical groups. With eitherapproach, the compounds that are selected to be added to the subcore maybe those that have been identified as subcore moieties derived fromother compounds that have exhibited desirable properties, or they may beof an uncharacterized or unselected nature.

As mentioned above, the aforementioned complex effects may be explainedby the possibility that pharmacological agents are binding to more thanone domain that is present in multiple copies in the target protein.Advantage of this can be taken on a broader scale than that described inthe present invention regarding subcore moieties by linkingpharmacological agents together that are too large to fit into a singledomain together. They may comprise a homodimer (or more) of identical orsimilar compounds, or they may comprise different agents. Althoughproteins such as LRP5 and LRP6 are frequently drawn as linear moleculeswith domains, akin to knots on a string, there may be flexing andbending of these proteins such that protein domains may be in closerphysical proximity than depicted in relevant diagrams. As such, amultimeric pharmacological agent that is formed by linking together twoor more previously selected pharmacological agents may allow forsituations where the binding of one agent to a high affinity domain willenhance the binding of a tethered second version of the agent to a loweraffinity domain, thereby creating new properties that would not beenjoyed by a monomeric agent at that concentration. Furthermore, sincethere may be compounds that optimally bind to different domains (seediscussion of IC15 and IIIC3 above) utility may also be found increating a multimeric compound that comprises two different moleculeswith their own specific affinities to potentially: a) increase theoverall affinity for the complex to the target; b) provide a wider rangeof targets that may be bound by the compound; and c) exhibit synergisticeffects. There may also be bifunctional binding to more than one proteinby a single multimeric pharmacological agent since dimerization andmultimerization of proteins is a common biological phenomenon that wouldprovide proximity between domains from different proteins.

The connection between Wnt signaling and inflammation is a complex issuewhere Wnt may be part of a number of disease processes such as pulmomaryfibrosis (Morrisey 2003 Am J Path 162; 1393-97; Pongracz and Stockley2006 Respiratory Research 7; 15), leukocyte inflammatory responses(Tickenbrock 2006 J Leuk Biol 79; 1306-1311) and diabetes (Figueroa etal. 2000). Increased levels of Wnt have also been seen in diseases suchas rheumatoid arthritis where it has been associated with increasedlevels of markers for inflammation such as IL-6, IL-8 and IL-15 in onestudy (Sen et al., 2000 Proc. Nat. Acad. Sci. (USA) 2791-2796) andTNF-α, IL- 1β and IL6 in another (Nakamura et al., 2005 Am J Path 167;97-105). In Nakamura et al., a direct connection between Wnt and thelatter set of inflammatory markers was shown by transfecting cells withWnt 7b and observing a significant increase in the level of all three ofthe markers. The opposite experimental analysis was carried out byGustafson and Smith (2006 J Biol Chem 281; 9507-9516) where treatment ofadipocytes with additional exogenous TNF-α increased Wnt expression andIL6 resulted in an increase of the apparent phosphorylation of frizzled,both events leading to a block in differentiation of the adipose cells.The effects of the Wnt pathway on adipogeneseis can also be seen wheretreatment with TNF-α resulted in the stabilization of β-catenin.(Cawthorn et al., 2007 Cell Death Differ 14; 1361-1373). This lattereffect could be reversed in a β-catenin knockout mouse where theblockage of adipogenesis by TNF-α was noticeably attenuated.

Rheumatoid arthritis is a disease that is marked by the presence of bothincreased Wnt and inflammatory cytokines. This is not surprising sincethe manifestations of this autoimmune disease involve bone reabsorptionas well as inflammation processes. A transgenic mouse has been developedas a model for rheumatoid arthritis in humans by transformation withhuman TNF, thereby replicating many of the features of the disease. Whenthis animal model was administered a Dkk-1 antibody, the result was theprevention of bone loss (Diarra et al. 2007 Nature Medicine 13;156-163). However, it was also found that there was an “uncoupling”where there was also no significant change in histopathologicalindications showing that a beneficial effect was only conveyed for partof the syndrome. Presumably, the anti-Dkk blocked repression by nativeDkk molecules and allowed increased Wnt expression to thereby amelioratethe bone loss problem. Since increased Wnt levels are associated withinflammation, it is not surprising that the inflammatory processcontinued in this study despite the treatment. In contrast, it wasdiscovered that the use of a small molecule selected for its ability tobind to the domain on LRP6 which is involved in Dkk binding gave asurprising and unanticipated event. In the animal model system used inExample 4, the pharmacological agent was able to duplicate the abilityof anti-Dkk to prevent bone loss but in contrast to the antibodyresults, the administration of the small molecule also led to areduction in the inflammation marker, TNF-α (see Example 4).

In addition, Li et al. described the use of anti-Dkk as a treatment forinflammatory processes in U.S. Patent Publication No. 2006/0127393. Thisapplication was mainly concerned with improvements in the nature of theanti-Dkk antibodies and there were no working examples provided fordemonstrating a reduction in inflammation by means of their antibody.Furthermore, in light of the work cited above by Diana et al., there isno evidence that the anti-Dkk antibody is capable of providing relief ofinflammation.

It is a further teaching of the present invention that agents that bindto LRP molecules, or to associated ligands or molecules, may haveindirect effects. For example, the binding of Kremen to Dkk and LRP5 andLRP6 is believed to lead to endocytosis of a ternary complex (reviewedin Rothbacher and Lemaire 2002 Nature Cell Biology 4; E172-E173) therebydecreasing the effective amount of LRP5 and LRP6 on the surfaces ofcells. As such, the inhibition of binding between Dkk and LRP5 and LRP6should result in a higher level of LRP5 and LRP6 remaining on thesurface of the cells. Conversely, pharmacological agents that increasebinding between LRP5 and LRP6 and Dkk should lead to increasedsequestration of LRP5 and LRP6 and a net decrease in its presence. Theseactions may influence the effects of any proteins that interact withLRP5 and LRP6, Dkk or Kremen and as discussed previously, these proteinsmay or may not be involved in Wnt signaling. The effects of such anincrease may also be complex in nature. For instance, it has been foundthat depending upon context, the effects of overexpression may bedifferent between LRP5 and LRP6. An overabundance of LRP5 has beenreported to lead to increased levels of β-catenin (Kato et al., J CellBiol 157; 303-314) and an overabundance of LRP6 has been described tolead to increased Wnt signaling (Liu et al., 2003 Molec and Cell Biol23; 5825-5835). However, Mi and Johnson (J Cell Biochem 95; 328-338)observed a difference between LRP5 and LRP6, where heightened levels ofLRP6 led to increased signals from the TCF/LEF marker, whereas LRP5 hadno effect. Although the baseline level of signaling was different, boththe LRP5 transfected cells as well as the LRP6 cells still showedevidence of increased signaling when Wnt was added.

Furthermore, different components of the Wnt system have differentfeedback loops that affect each other's level of transcription. Forexample, the use of siRNA to knock down the amount of Dkk provides atransient increase in Wnt activity, but this is counterbalanced by thepresence of motifs in the promoter for Dkk leading to upregulation oftranscription from the Dkk. The amount of Dkk activity may be equal tothe initial amount or it may be higher or lower, depending upon theamount of transcription carried out. This may provide at least a partialexplanation for the results of the anti-Dkk antibody discussed above.

Pharmacological agents found capable of binding to LRP or to an LRPassociated protein may find use with other processes that have beenfound associated with the Wnt pathway. For example, it has been recentlydiscovered that Wnt activity has been linked to hair follicle formation(Aandl et al., 2002 Developmental Cell 2; 643-653; Sick et al., 2006Science 1447-1450) and as such some of the compounds of the presentinvention may be used to ameliorate hair loss problems. In addition, agroup of proteins called matrix metalloproteinases (mmps) have beenfound to be associated with skin biology during inflammatory matrixremodeling neovascularization, wound healing and malignanttransformation as well as less serious conditions such as acne(Papakonstantinou et al., J Invest Dermatol 125; 673-684). Some of thesemmps, including MMP2, MMP3, MMP7 and MMP9, have been described astargets of the Wnt signaling pathway (Tamamura et al., 2005 J Bioi Chem280; 19,185-19,195). The mmps may also illustrate a connection betweenWnt activity and inflammation since treatment of breast cancer cellswith Wnt5a led to induction of MMP7 which is known to release TNF-α.(Pukrop et al., 2006 Proc. Nat. Acad. Sci (USA) 103; 5454-5459).Therefore, certain compounds of the present invention may possesscurative processes for disease conditions associated with mmps.

The terms “immune modulation” should be understood to mean themodification of one or more components of the immune system to eitherenhance or inhibit the activity or amount of that component orcomponents. Modulation may also include a simultaneous enhancement ofone or more components accompanied by inhibition of one or more othercomponents.

The terms “immune disorders” are diseases involving the immune systemthat can include but not be limited to allergies, autoimmune diseases,immune complex diseases, immunodeficiency diseases and cancers of theimmune system.

The term “autoimmune diseases” may include but not be limited to Acutedisseminated encephalomyelitis, Addison's disease, Ankylosingspondylitisis, Antiphospholipid antibody syndrome, Aplastic anemia,Autoimmune hepatitis, Autoimmune Oophoritis, Coeliac disease, Crohn'sdisease, Diabetes mellitus, Gestational pemphigoid, Goodpasture'ssyndrome, Grave's disease, Guillan-Barre syndrome, Hashimoto's disease,Idiopathic thrombocytopenic purpura, Lupus erythematosus, Multiplesclerosis, Myasthenia gravis, Opsoclonus myoclonus syndrome, Opticneuritis, Ord's thyroiditis, Pemphigus, Pernicious anemia,Polyarthritis, Primary biliary cirrhosis, Rheumatoid arthritis, Reiter'ssyndrome, Sjogren's syndrome, Takayasu's arteritis, Warm autoimmunehemolytic anemia, and Wegener's granulomatosis.

The term “chronic inflammatory diseases” may include but not be limitedto Tuberculosis, Chronic cholecystitis, Bronchiectasis, ulcerativecolitis, silicosis and other pneumoconiosis as well as the above listedautoimmune diseases.

The term “small molecule” is means a non-peptide molecule of 10,000 orless molecular weight.

The terms “administration of” or “administering a” compound should beunderstood to mean providing a compound of the invention to theindividual in need of treatment in a form that can be introduced intothat individual's body in a therapeutically useful form andtherapeutically useful amount, including, but not limited to: oraldosage forms, such as tablets, capsules, syrups, suspensions, and thelike; injectable dosage forms, such as IV, IM, or IP, and the like;transdermal dosage forms, including creams, jellies, powders, orpatches; buccal dosage forms; inhalation powders, sprays, suspensions,and the like; and rectal suppositories. The terms “therapeuticallyeffective amount” means the amount of the subject compound that willelicit the biological or medical response of a tissue, system, animal orhuman that is being sought by the researcher, veterinarian, medicaldoctor or other clinician. As used herein, the term “treatment” refersto both to the treatment and to the prevention or prophylactic therapyof the mentioned conditions, particularly in a patient who ispredisposed to such disease or disorder.

The term “treating” in its various grammatical forms in relation to thepresent invention refers to preventing, (i.e., chemoprevention), curing,reversing, attenuating, alleviating, minimizing, suppressing or haltingthe deleterious effects of a disease state, disease progression, diseasecausative agent (e.g., bacteria or viruses) or other abnormal condition.For example, treatment may involve alleviating a symptom (i.e., notnecessary all symptoms) of a disease or attenuating the progression of adisease. Because some of the inventive methods involve the physicalremoval of the etiological agent, the artisan will recognize that theyare equally effective in situations where the inventive compound isadministered prior to, or simultaneous with, exposure to the etiologicalagent (prophylactic treatment) and situations where the inventivecompounds are administered after (even well after) exposure to theetiological agent.

The term “LRP ligand” is a protein involved in a protein-proteininteraction with at least one member of the LRP receptor family. Ligandsinclude proteins, lipoproteins, proteinases, proteinase inhibitorcomplexes, ECM proteins, bacterial toxins, viruses and variousintracellular and extracellular proteins. Examples of ligands that areknown to interact with LRPS and LRP6 include Wnt, Sclerostin (SOST),Wise, DKK and Frizzled (Frz).

EXAMPLES

Examples provided are intended to assist in a further understanding ofthe invention. Particular materials employed, species and conditions areintended to be further illustrative of the invention and not limited ofthe reasonable scope thereof.

Example 1

Effects of Gallic Acid and Digallic Acid on Wnt and Dkk suppression ofWnt.

This experiment was carried out as previously described in U.S. PatentPublication No. 2005/0196349 using Gallic Acid, a small molecule thatrepresents a partial constituent of the IIIC3 molecule as well asDigallic Acid, which represents a dimeric form of Gallic Acid. Thestructures of Gallic Acid and Digallic Acid are provided below:

As seen in FIG. 2A, the small molecule derivative of IIIC3 is capable ofproviding protection against Dkk suppression when present at 30 mM. Inthis experiment, the Digallic Acid completely blocked Dkk at even thelowest (1.2 mM) value tested.

Example 2

Effects of Digallic Acid on Wnt and Dkk suppression of Wnt. Thisexperiment was carried out as described above except that a lower rangeof drug dosage was used as compared to Example 1. In FIG. 3A, there isessentially no effect upon either Wnt activity or suppression of Wntactivity by Dkk when up to 3.3 mM Gallic Acid was present (similar towhat was seen with Example 1, FIG. 2A). In contrast, FIG. 3B shows thatmodest effects upon Wnt activity at the higher (1.1 and 3.3 uM) dosagesand a dose dependent effect upon inhibition of Dkk suppression showingthat the dimeric form is much more potent than the monomeric form. BothFIGS. 2 and 3 indicate that nearly 30 times as much Gallic Acid had tobe present to achieve the same effect as the dimeric Gallic Acid.

Example 3

Stimulatory Effects of Enzo IIC8 (shown below) on Alveolar New BoneFormation in a tooth extraction model.

A root extraction model (Lin et al., 1994 Anat Record 240; 492-506) wasused to determine whether IIC8 (described in U.S. Patent Publication No.2005/0196349) is able to stimulate new bone formation. The boneregeneration process following tooth extraction is a complex phenomenonthat involves wound healing, as well as bone formation. Briefly, theinitial coagulum is followed by the formation of woven bone, lamellarbone, bone marrow, and cortical bone. At the cellular level this processinvolves induction and regulation of growth of several distinct celltypes, as well as differentiation of stem cells into several cell types.The point of the experiment described below was to determine whether adrug could accelerate the process of bone growth without adverselyaffecting the end product of the process.

Procedure: 10 week old Sprague Dawley rats (˜300 gram body weightobtained from Taconic Farms, Germantown, Pa.) were anesthetized. Theythen underwent extraction of left and right first maxillary molarsfollowed by filling of the empty tooth sockets with gel foam. Theanimals were than treated both topically and systemically with the testcompound. In Group A, 8 rats were injected with 10 μl A of 5mg/ml IIC8dissolved in PBS. In Control Group B, 8 rats were injected with 10 μl ofPBS. At approximately 12 hr intervals the animals were injected withadditional 10 ml aliquots of IIC8 (Group A), or PBS (Group B). At thesame time, the animals were also injected IP with 1 ml of IIC8 (GroupA), or with 1 ml of PBS (Group B). This treatment was carried out infive day cycles, followed by two days of rest, for a total duration of 3weeks. At 7 days intervals, two animals from each group were sacrificedand their maxillae were excised, fixed, and decalcified. Afterdehydration, the specimens were sectioned along the molars in amesio-distal plane followed by staining with hematoxylin and eosin.

Results: As shown in FIG. 4A, after one week of treatment, the IIC8treated animals already exhibit a large number of osteoblast cells,indicating significant osteoblast differentiation/proliferation andosteoblastic activity. Osteoclast cells are also seen, indicating thatbone remodeling and reconstruction is in process with a notable amountof new bone being deposited. In contrast, very few osteoblast orosteoclast cells are found in the control group (FIG. 4).

After 2 weeks of treatment, an overwhelming number of osteoblast cellswere found inside the extracted tooth sockets of the IIC8 treatedanimals, with a decreased number of osteoclast cells relative to thesamples from week one, indicating an extremely high level of anabolicactivity. There was a significant amount of mineralized new bone formedat this stage.

Animals in the PBS control group also had osteoblast cells after twoweeks, which confirms that the remodeling process is triggered by thetooth extraction and thus is active, though to a lesser extent, even inthe absence of any drug treatment.

After 3 weeks of treatment, the specimens showed reduced osteoblasticactivities in both IIC8 and PBS groups. However, in the IIC8 treatedgroup, there was a significant amount of mineralized new bone throughoutthe socket. In contrast, new bone formation was seen only in a few smallareas of the control group.

Conclusion: The tooth extraction model, a standard model of boneformation and remodeling, shows that IIC8 significantly stimulates bothprocesses relative to the untreated controls. Thus IIC8 can be utilizedas an agent that promotes osteogenesis and upregulates anabolicactivity. Additionally, the concomitant topical and systemicadministration proved free of undesired (toxic) effects, which providesconsiderable leeway in the design of a therapeutic regimen.

Example 4

Potency of Enzo IIC8 in the prevention of LPS-induced periodontal boneloss.

IIC8 was tested in an animal model of periodontitis (Miyauchi et al.2001 Histochem Cell Biol. 116:57-62) that was used to evaluate cytokineproduction in rat molar gingival periodontal tissues after topicalapplication of lipopolysaccharide (LPS). LPS is a complex glycolipidthat represents a major component of the outer membrane of Gram-negativebacteria, which are well established etiological agents ofperiodontitis.

Remarkably, with regard to periodontitis, LPS alone can mimic the effectof a bacterial infection, by establishing an inflammatory condition thateventually leads to periodontal tissue destruction. Thus, the model iswell suited to test for drugs that help prevent bone loss elicited bymassive inflammation.

Procedure: Sprague Dawley rats ˜300 gram body weight obtained fromTaconic Farms, Germantown Pa.) were treated with LPS, or with PBS, byinjection into the maxillary labial and palatal gingival between firstand second upper molars on both sides. The injections were repeated twomore times on an every other day basis, for a total of three treatments.

Three groups were investigated:

Group A: PBS-treated, 10 animals

Group B: LPS-treated, 10 animals

Group C: LPS- and IIC8-treated, 12 animals

For Group C, 1 ml of 5mg/m1 IIC8 was administered per os daily startingfrom three days prior to the initial LPS injection for a total durationof 10 days.

At the conclusion of treatments, animals were euthanized and theirmaxillary jaws excised and defleshed. The defleshed jaws were thensoaked in 0.2N NaOH for 5 min at room temperature to remove theremaining soft tissue and analyzed under a dissection microscope.

Results: Inspections of the jaws under dissection microscope showed thatanimals treated with IIC8 had significantly more alveolar bone thanthose in the LPS-only control group. FIGS. 5A is a macroscopic image ofthe palatal sides of the maxillary jaws. The control with the LPSadministration showed severe bone resorption with root furcationexposure, demonstrating the major destructive impact LPS has in thisanimal model system. On the other hand, there is very limited loss ofthe alveolar bone in the group of animals that have been administeredcompound IIC8 as well as the LPS, showing a highly protective effect bythis compound. Although it can be seen by the naked eye that the IIC8conferred beneficial effects in this system, a duplicate experiment wascarried out and measurements were made between the cementoenameljunctions (CEJ) and the alveolar crests to obtain numerical data. Asecond experiment was also carried out and quantitative measurementswere taken. The defleshed jaws were stained with Leoffler's methyleneblue in order to identify the cemento-enamel junction (CEJ) as areference point to measure bone height. Histological analysis clearlyshowed significant bone resorption and root furcation in the LPS-treatedanimals, and little bone resorption in the LPS plus SMTC-treatedanimals. Linear measurements from the CEJs to the alveolar bone crestshowed a mean bone loss of 0.94±0.08 mm in LPS-treated animals;0.59±0.04 mm in LPS plus SMTC- treated animals; and 0.54±0.04 in controlanimals. There were statistically significant differences between theLPS group and the LPS plus SMTC group (p =0.00006) and between thecontrol group and LPS group (p=0.00003). As an indicator of protection,there was no significant difference between the control group and theLPS plus SMTC group (p 0.18). These data clearly show that SMTC protectsagainst bone resorption in an animal model of endotoxin-induced boneloss. This SMTC may represent an attractive potential new class oftherapeutic agents for clinical use.

Quantitative results were also obtained by measurements of TNF-α, whichas described previously is a major marker for inflammatory processes.The results of this assay were as follows:

Group A: PBS-treated 59.7 Group B: LPS-treated 102.9 Group C: LPS- andIIC8-treated 65.2

It should be pointed out that although the differences between Group Aand B as well as between Group B and C were highly significant (P valuesof 0.0001 for each), the difference between Groups A and C was notconsidered to have significance (tailed P value equals 0.3479), i.e. theuntreated controls and the subjects treated with II1C8 in addition tothe LPS are statistically undistinguishable. This shows that in additionto either preventing bone loss or compensating for its loss, the IIIC8compound was also able to reduce inflammation that had been the primarycause of the disease process of the animal model. It also serves as anexample that the compounds of the present invention may have utility inwound healing processes.

Example 5

Characterization of DKK(−/−) Mice

The complexity of the interplay between LRP5 and LRP6, Dkk andinflammation was examined by testing the effects of inflammatoryresponses in knockout mice that had Dkk2 eliminated (this mutation waspreviously described in U.S. Patent Publication No. 2005/0261181). Alsotested were heterozygous Dkk (Dkk2 +/0) mice where due to gene dosageeffects there should be lower intrinsic levels of Dkk present.

Results: Various parameters are shown in FIG. 6 and FIG. 7 where majordifferences may be seen for TNF-α levels (FIG. 6A), blood glucose levels(FIG. 6B) and Adiponectin (FIG. 7).

1. A method for promoting wound healing, comprising: administering to ahuman patient in need of wound healing a therapeutically effectiveamount of a compound selected from the group consisting of:


2. The method of claim 1, wherein the human patient has Diabetesmellitus.
 3. The method of claim 1, wherein the compound is


4. The method of claim 3, wherein the human patient has Diabetesmellitus.
 5. The method of claim 1, wherein the compound is


6. The method of claim 5, wherein the human patient has Diabetesmellitus.
 7. The method of claim 1, wherein the compound is


8. The method of claim 7, wherein the human patient has Diabetesmellitus.